The present invention relates to oligomers containing aminooxy linkages and methods of using such oligomers. More particularly, the oligomers of the present invention are used for investigative and therapeutic purposes.
It has been recognized that oligonucleotides can be used to modulate mRNA expression by a mechanism that involves the complementary hybridization of relatively short oligonucleotides to mRNA such that the normal, essential functions of these intracellular nucleic acids are disrupted. Hybridization is the sequence-specific base pair hydrogen bonding of an oligonucleotide to a complementary RNA or DNA.
For use in diagnostics, and as research reagents and as therapeutics, the ability of an oligonucleotide to bind to a specific DNA or RNA with fidelity is an important factor. The relative ability of an oligonucleotide to bind to complementary nucleic acids is compared by determining the melting temperature of a particular hybridization complex. The melting temperature (Tm), a characteristic physical property of double helices, is the temperature (in xc2x0C.) at which 50% helical versus coil (unhybridized) forms are present. Tm is measured by using UV spectroscopy to determine the formation and breakdown (melting) of hybridization. Base stacking, which occurs during hybridization, is accompanied by a reduction in UV absorption (hypochromicity). Consequently, a reduction in UV absorption indicates a higher Tm. The higher the Tm, the greater the strength of the binding of the nucleic acid strands. Therefore, oligonucleotides modified to hybridize with appropriate strength and fidelity to its targeted RNA (or DNA) are greatly desired for use as research reagents, diagnostic agents and as oligonucleotide therapeutics.
Various substitutions have been introduced in the base and sugar moieties of the nucleosides of oligonucleotides. The inclusion of certain of these substitutions has resulted in improvements in the resulting oligonucleotide. One such useful improvement is an increase in the nuclease resistance of the oligonucleotides by the introduction of 2xe2x80x2-substituents such as alkoxy, allyloxy, and aminoalkyl groups.
Ikehara et al. (European J. Biochem., 1984, 139, 447) have reported the synthesis of a mixed octamer containing one 2xe2x80x2-deoxy-2xe2x80x2-fluoroguanosine residue or one 2xe2x80x2-deoxy-2xe2x80x2-fluoroadenine residue. Guschlbauer and Jankowski (Nucleic Acids Res, 1980, 8, 1421) have shown that the contribution of the 3xe2x80x2-endo increases with increasing electronegativity of the 2xe2x80x2-substituent. Thus, 2xe2x80x2-deoxy-2xe2x80x2-fluorouridine contains 85% of the C3xe2x80x2-endo conformer.
Furthermore, evidence has been presented which indicates that 2xe2x80x2-substituted-2xe2x80x2-deoxyadenosine polynucleotides resemble double-stranded RNA rather than DNA. Ikehara et al. (Nucleic Acids Res., 1978, 5, 3315) have shown that a 2xe2x80x2-fluoro substituent in poly A, poly I, or poly C duplexed to its complement is significantly more stable than the ribonucleotide or deoxyribonucleotide poly duplex as determined by standard melting assays. Ikehara et al. (Nucleic Acids Res., 1978, 4, 4249) have shown that a 2xe2x80x2-chloro or bromo substituent in poly(2xe2x80x2-deoxyadenylic acid) provides nuclease resistance. Eckstein et al. (Biochemistry, 1972, 11, 4336) have reported that poly(2xe2x80x2-chloro-2xe2x80x2-deoxy-uridylic acid) and poly(2xe2x80x2-chloro-2xe2x80x2-deoxycytidylic acid) are resistant to various nucleases. Inoue et al. (Nucleic Acids Res., 1987, 15, 6131) have described the synthesis of mixed oligonucleotide sequences containing 2xe2x80x2-OMe substituents on every nucleotide. The mixed 2xe2x80x2-OMe-substituted oligonucleotide hybridized to its RNA complement as strongly as the RNA-RNA duplex which is significantly stronger than the same sequence RNA-DNA heteroduplex (Tms, 49.0 and 50.1 versus 33.0 degrees for nonamers). Shibahara et al. (Nucleic Acids Res., 1987, 17, 239) have reported the synthesis of mixed oligonucleotides containing 2xe2x80x2-OMe substituents on every nucleotide. The mixed 2xe2x80x2-OMe-substituted oligonucleotides were designed to inhibit HIV replication.
It is believed that the composite of the hydroxyl group""s steric effect, its hydrogen bonding capabilities, and its electronegativity versus the properties of the hydrogen atom is responsible for the gross structural difference between RNA and DNA. Thermal melting studies indicate that the order of duplex stability (hybridization) of 2xe2x80x2-methoxy oligonucleotides is in the order of RNA-RNA greater than RNA-DNA greater than DNA-DNA.
U.S. Pat. No. 5,013,830, issued May 7, 1991, discloses mixed oligonucleotides comprising an RNA portion, bearing 2xe2x80x2-O-alkyl substituents, conjugated to a DNA portion via a phosphodiester linkage. However, being phosphodiesters, these oligonucleotides are susceptible to nuclease cleavage.
European Patent application 339,842, filed Apr. 13, 1989, discloses 2xe2x80x2-O-substituted phosphorothioate oligonucleotides, including 2xe2x80x2-O-methylribooligonucleotide phosphorothioate derivatives. This application also discloses 2xe2x80x2-O-methyl phosphodiester oligonucleotides which lack nuclease resistance.
European Patent application 260,032, filed Aug. 27, 1987, discloses oligonucleotides having 2xe2x80x2-O-methyl substituents on the sugar moiety. This application also makes mention of other 2xe2x80x2-O-alkyl substituents, such as ethyl, propyl and butyl groups.
International Publication Number WO 91/06556, published May 16, 1991, and U.S. Pat. No. 5,466,786 discloses oligomers derivatized at the 2xe2x80x2 position with substituents, which are stable to nuclease activity. Specific 2xe2x80x2-O-substituents which were incorporated into oligonucleotides include ethoxycarbonylmethyl (ester form), and its acid, amide and substituted amide forms.
European Patent application 399,330, filed May 15, 1990, discloses nucleotides having 2xe2x80x2-O-alkyl substituents.
International Publication Number WO 91/15499, published Oct. 17, 1991, discloses oligonucleotides bearing 2xe2x80x2-O-alkyl, -alkenyl and -alkynyl substituents.
Martin discloses certain nucleosides and oligonucleotides prepared therefrom that include 2xe2x80x2-methoxyethoxy, 2xe2x80x2-methoxy(tris-ethoxy) and other substituents. Helvetica Chimica Acta, 78, 1995, 486-504. Oligonucleotides containing nucleoside substituted with either the 2xe2x80x2-methoxyethoxy and 2xe2x80x2-methoxy(tris-ethoxy)substituents exhibited improved hybridization as judged by increase in Tm.
The expanding use of mixed-phase hydridization assays for the detection of specific nucleic acid sequences has made covalent immobilization of oligonucleotides to solid supports an object of increasing interest. See, Lund et al., Nucleic Acids Res., 1988, 16, 10861; Saiki et al., Proc. Natl. Acad. Sci. U.S.A., 1989, 86, 6230; Albretsen et al., Anal. Biochem., 1990, 189, 40; Erout et al., Bioconjugate Chem., 1996, 7, 568; Yershov et al., Proc. Natl. Acad. Sci. U.S.A., 1996, 93, 4913; and Hakala et al., Bioconjugate Chem., 1998, 9, 316.) Although the oligonucleotide probes may be assembled in situ on the support employed in the assay (Maskos and Southern, Nucleic Acids Research, 1992, 20, 1679; Pirrung and Bradley, J. Org. Chem., 1995, 60, 6270; and Cohen et al., Nucleic Acids Res., 1997, 25, 911), post-synthetic attachment of purified oligonucleotide conjugates to the support may still be advantageous for some applications. A variety of methods for tethering oligonucleotides to solid supports have been reported. Most of the methods are based on reactions of 5xe2x80x2-aminoalkyl conjugates of oligonucleotides with various functional groups on the support. For example, oligonucleotides bearing a 5xe2x80x2-terminal amino function have been attached to: (i) to carboxyalkylated polymer supports by carbodiimide assisted acylation (Ghosh and Musso, Nucleic Acids Res., 1987, 15, 5353; Zhang et al., Nucleic Acids Res., 1991, 19, 3929); (ii) amino-alkylated polymer supports by activation with 2,4,6-trichloro-1,3,5-triazine and subsequent displacement of one of the remaining chloro substituents with a resin bound amino group (Van Ness et al., Nucleic Acids Res., 1991, 19, 3345); (iii) aldehyde-derivatized surfaces by reductive amination (Timofeev et al., Nucleic Acids Res., 1996, 24, 3142); and (iv) phenyl-diisothiocyanate activated (Guo et al., Nucleic Acids Res., 1994, 22, 5456) or epoxide-derivatized glass (Lamture et al., Nucleic Acids Res., 1994, 22, 2121) surfaces by direct nucleophilic substitution. 5xe2x80x2-Phosphorylated oligonucleotides have been immobilized to aminoalkylated supports by carbodiimide-assisted phosphoramidate coupling (Ghosh and Musso, Nucleic Acids Res., 1987, 15, 5353), and 5xe2x80x2-mercapto-functionalized oligonucleotides to mercaptoalkyl supports by disulfide formation (Bischoff et al., Anal. Biochem., 1987, 164, 336). The latter oligonucleotides have also been successfully immobilized onto iodoacetamido-derivatized supports by nucleophilic xcex1-substitution (O""Donnell et al., Anal. Chem., 1997, 69, 2438).
Oligonucleotides bearing a 5xe2x80x2-terminal aldehyde function have been attached to aminoalkylated supports by reductive amination (Timofeev et al., Nucleic Acids Res., 1996, 24, 3142), and to latex microspheres bearing hydrazine residues (Kremsky et al., Nucleic Acids Res., 1987, 15, 2891). 2,4,6-Trichloro-1,3,5-triazine activation and disulfide bond formation have also been exploited in immobilization of 3xe2x80x2-amino and 3xe2x80x2-mercapto functionalized oligonucleotides, respectively (Hakala et al., Bioconjugate Chem., 1997, 8, 232). Homopolymer-tailed oligomers have been attached to a nylon membrane by UV irradiation (Saiki et al., Proc. Natl. Acad. Sci. U.S.A., 1989, 86, 6230).
The use of nucleic acid derived probes as diagnostic tools has seen widespread application. Typically, these probes are short oligonucleotides designed to bind to complementary target nucleic acids and have found applications in the detection of bacterial, viral and fungal infections, PCR reaction product, the study of polymorphisms, expression levels and genetic diseases. Their promise lies in the exquisite specificity and sensitivity of the hybridization reaction wherein the alteration or deletion of even one base leads to a dramatic change in the binding of the probe and target. Detection of the hybridized probe-target complex therefore allows for the identification of the nature and sequence of the target nucleic acid.
Diagnostic hybridization probes have been used in the art in homogenous conditions, such as in solution, and in heterogenous conditions, such as in mixed-phase hybridization assays. Mixed-phase hybridization assays typically utilize either the target or, more commonly, the probe in an immobilized form. Immobilization and mixed-phase hybridization have the primary advantage of ease of separation of the hybridized complex from excess reagents.
To further improve the utility of such diagnostic assays, arrays of nucleic acids have also been used. Such arrays of nucleic acid probes have provided advantages of parallel screening of nucleic acid targets, miniaturization, multiplexing and automation of such diagnostic procedures. Detection methods that have been commonly used in oligonulceotide probe diagnostic assays include radioactivity, fluorescent reporters, optical wave guides and mass spectrometry.
Such arrays have been used to sequence large nucleic acids via sequencing by hybridization (SBH). Typically, the probes are immobilized or a set of unknown targets are immobilized and sequentially hybridized with either the target nucleic acids or oligonucleotide probes, respectively. Tiling arrays have been widely used to systematically identify the base at each position of an unknown nucleic acid (Chee et al., Science, 274 (1996) 610-614). Tiling arrays have been used in polymorphism studies of the HIV Protease gene (Kozal et al., Nat. Med., 2 (1996) 753-9), and for mutation studies of hereditary breast cancer gene BRCA1 (Hacia et al., Nucleic Acids Res., 26 (1998) 3865-66) and cystic fibrosis (Cronin et al., Human Mut., 7 (1996) 244-55). Oligonucleotide arrays have also been used in combination with enzymes such as ligases and polymerases to provided enhanced methods of mutation analysis.
It has been recognized that oligonucleotides having reactive groups capable of reacting with specific reporter groups, ligands, cell surface targeting agents, mRNA target cleavage agents, solid supports, nylon membranes, silicon chips, glass plates, glass slides and microparticles are of great importance in the development of oligonucleotides that are useful as research reagents, diagnostic agents and therapeutic agents, as well as in DNA arrays.
In accordance with the present invention, oligomers containing aminooxy linkages are provided. Preferred compositions include oligomers comprising a plurality of nucleotide units of the structure: 
wherein:
Bx is a purine or pyrimidine heterocyclic base;
each T1 and T2 is, individually, OH, a protected hydroxyl, a nucleotide, a nucleoside or an oligonucleotide;
T3 is H, OH, a protected hydroxyl or a sugar substituent group;
said oligomer further comprising at least one group, R, therein; said R group occurring at the 5xe2x80x2-end, the 3xe2x80x2-end, in lieu of at least one T3 or as a substituent on at least one Bx; said R group having one of the formulas: 
wherein:
each Z is, independently, a single bond, 0 or a phosphate;
each Q is, independently, H, C1-C10 alkyl or a nitrogen protecting group;
each T0 is, independently, a bond or a linking moiety;
each L is, independently, a chemical functional group, a conjugate group or a solid support material;
or Q, T0 and L, together, are a chemical functional group;
each m is, independently, an integer from 1 to about 10; and
each n is, independently, an integer from 1 to about 6.
In a preferred embodiment, R is a conjugate group. In another preferred embodiment, R is a solid support material. In yet another embodiment, R is a chemical functional group.
In a preferred group of compounds the conjugate group is a contrast reagent, a cleaving agent, a cell targeting agent, polyethylene glycol, cholesterol, phospholipid, biotin, phenanthroline, phenazine, phenanthridine, anthraquinone, acridine, fluorescein, rhodamine, coumarin, pyrene, retinal or a cyanine dye. In another preferred group of compounds the solid support material is microparticles or CPG.
The present invention also provides methods for diagnosing the presence of nucleic acids in a sample comprising the steps of attaching an oligonucleotide containing an aminooxy linkage to a solid support material; labeling the oligonucleotide with a marker to form a labeled oligonucleotide; treating the labeled oligonucleotide with a target oligonucleotide to form a hybridization mixture; detecting binding of the labeled oligonucleotide with the target oligonucleotide in the hybridization mixture; and determining the amount of labeled oligonucleotide bound to the target oligonucleotide.
The present invention also provides another method of diagnosing the presence of nucleic acids in a sample comprising the steps of attaching an oligonucleotide containing an aminooxy linkage to a solid support material; treating the oligonucleotide with a target oligonucleotide to form a hybridization mixture, wherein the target oligonucleotide is labeled with a marker; detecting binding of the oligonucleotide with the target oligonucleotide in the hybridization mixture; and determining the amount of oligonucleotide bound to the target oligonucleotide.
In a preferred embodiment the solid support material comprises an aldehyde group. In another preferred embodiment the solid support material comprises an epoxy group. In yet another preferred embodiment the solid support material is microparticles. It is preferred that the marker be a fluorescent marker. It is also preferred that the labeling step comprise forming a lanthanide chelate with the oligonucleotide. It is further preferred that the detecting step comprise measuring the fluorescence emission of the hybridization mixture.